Deciphering Transcription in Cryptosporidium parvum: Polycistronic Gene Expression and Chromatin Accessibility

Once considered rare in eukaryotes, polycistronic mRNA expression has been identified in kinetoplastids and, more recently, green algae, red algae, and certain fungi. This study provides comprehensive evidence supporting the existence of polycistronic mRNA expression in the apicomplexan parasite Cryptosporidium parvum. Leveraging long-read RNA-seq data from different parasite strains and using multiple long-read technologies, we demonstrate the existence of defined polycistronic transcripts containing 2-4 protein encoding genes, several validated with RT-PCR. Some polycistrons exhibit differential expression profiles, usually involving the generation of internal monocistronic transcripts at different times during development. ATAC-seq in sporozoites reveals that polycistronic transcripts usually have a single open chromatin peak at their 5-prime ends, which contains a single E2F binding site motif. Polycistronic genes do not appear enriched for either male or female exclusive genes. This study elucidates a potentially complex layer of gene regulation with distinct chromatin accessibility akin to monocistronic transcripts. This is the first report of polycistronic transcription in an apicomplexan and expands our understanding of gene expression strategies in this medically important organism. SignificanceCryptosporidium parvum is a parasite that causes human gastrointestinal disease, profoundly impacting infants and immunocompromised individuals worldwide. C. parvum isolation, culturing, cloning, and its compact 9.2 Mb genome pose challenges for research. We have discovered that, unlike most eukaryotes, which express one gene per mRNA, C. parvum exhibits widespread polycistronic transcription, whereby 10% of its protein encoding genes are expressed as multiple genes per single mRNA molecule. Mining the published C. parvum single-cell atlas with proxy sequences for polycistronic expression, we observed patterns of polycistron expression that vary with developmental stages, suggesting a role in the parasites lifecycle. By illuminating this unique aspect of C. parvum biology, our findings provide novel insights into Cryptosporidium gene expression.